Identification And Functional Analysis Of REVEILLEs Transcription Factors Regulating The Circadian Accumulation Of Anthocyanin In Pear Peel

Pear(Pyrus L.)belongs to the family of Rosaceae and is one of the major fruit trees cultivated both at home and abroad.The color of pear peel can be green,yellow,brown,and red,among which the red pear is favored by consumers because of its bright color and rich anthocyanin.The accumulation of anthocyanin in plants exhibits diurnal oscillations,and the circadian regulation of anthocyanin biosynthesis in most fruit trees such as pear is still unclear.The ‘Hongzaosu'(Pyrus cv.‘Hongzaosu')pear peel was used as main material in this study.The ‘Red Silk'(Pyrus communis L.cv.‘Red Silk'),‘Red Bartlett'(Pyrus communis L.cv.‘Red Bartlett'),‘Red Palacer'(Pyrus communis L.cv.‘Red Palacer'),and‘Zaosu'(Pyrus cv.‘Zaosu')were used as auxiliary test materials in this study.Two MYBrelated transcription factors,PbREVEILLE1 b and PbREVEILLE6(PbRVE1 b and PbRVE6),as well as their downstream transcription factor,PbbHLH2,were identified and investigated for their function in regulating diurnal oscillations of anthocyanin accumulation in pear.This study can provide a new theoretical basis and reference for circadian regulation of anthocyanin biosynthesis and pear fruit coloring.The main results are as follows:1.The anthocyanin accumulation and the expression pattern of anthocyanin biosynthesis structural genes in pear showed regular diurnal variation.The anthocyanin content of ‘Hongzaosu' pear peel was mainly accumulated at night,and the anthocyanin content in the daytime increased firstly and then decreased,the net accumulation of anthocyanin in the daytime was approach to zero.In addition,the expression levels of anthocyanin biosynthesis structural genes(Pb DFR,Pb ANS and Pb UFGT)in pericarp increased rapidly at night,but decreased after sunrise,and maintained a low expression levels in the daytime.The expression patterns of anthocyanin biosynthesis structural genes showed regular diurnal alternation in pear.However,the expression levels of the typical Pb MYBs transcription factors Pb MYB9,Pb MYB10 and Pb MYB10 b did not show this phenomenon.Therefore,these three factors are not the key regulators in diurnal biosynthesis of anthocyanin.2.The expression levels of anthocyanin biosynthesis structural genes were correlated with the expression levels of PbRVEs genes.The expressions of PbRVEs were diurnal.The expression levels of PbRVE1 a,PbRVE1 b,PbRVE7 and PbRVE8 reached a peak at dawn and then droped until sunset.This variation repeated itself on a 24-hour cycle.The expression levels of PbRVE3 and PbRVE6 increased at first and then decreased during the daytime,and this variation also repeated itself on a 24-hour cycle.There was a positive correlation between the expression levels of PbRVE1 b transcription factor and the expression levels of anthocyanin biosynthesis structural genes in the peel of ‘Hongzaosu'.The expression analysis showed that the PbRVEs expressed at different times.PbRVE1 a,PbRVE1 b,PbRVE7 and PbRVE8 were highly expressed at night,while PbRVE3 and PbRVE6 were mainly expressed in the daytime.3.PbRVE1b and PbRVE6 promoted the expression of structural genes and the anthocyanin biosynthesis in pear peel.Overexpression of PbRVE1 b and PbRVE6 significantly increased the accumulation of anthocyanin and the expression of structural genes in ‘Zaosu' peel.The virus-mediated gene silencing system was used to silence PbRVE1 b in pear peel,and the peel could not be colored,which further indicated that PbRVE1 b is a key transcription factor for promoting anthocyanin biosynthesis in pear peel.Although PbRVE6 promoted the accumulation of anthocyanin,the peel returned to red and the content of anthocyanin did not siginificantly changed after silencing this gene.This indicated that the role of PbRVE6 in promoting the anthocyanin accumulation could be complemented by other transcription factors in pear.The results of yeast one-hybrid(Y1H)and dual-luciferase analysis showed that PbRVE1 b can promote the anthocyanin biosynthesis by binding Pb DFR and Pb ANS promoters and activating their transcriptional activities.PbRVE6 can bind to the promoter of Pb ANS and Pb UFGT and activate their transcriptional activities to promote the anthocyanin biosynthesis.4.PbbHLH2 promotes the anthocyanin biosynthesis of pear peel and is regulated by PbRVE1 b and PbRVE6.Five IIIf subgroup PbbHLHs were screened by phylogenetic analysis.Expression pattern of PbbHLHs showed that the expression level of PbbHLH2 was regular diurnal changes.The expression of PbRVE1 b and PbRVE6 could affect the expression level of PbbHLH2.In addition to the basic-helix-loop-helix domain,there was also a MYB-interacting region(MIR)domain in PbbHLH2 protein through alignment analysis.The transient expression analysis showed that overexpression of PbbHLH2 increased the anthocyanin accumulation and promoted the expression levels of anthocyanin biosynthesis structural genes in pear peel.The peel could not be colored when PbbHLH2 was silenced in pear peel by virus-madiated gene silencing system.This result indicated that PbbHLH2 is a key transcription factor in anthocyanin biosynthesis in pear peel.PbbHLH2 interacted with Pb MYB9 or Pb MYB10 to form a complex through the MIR-domain,and the complex promoted the biosynthesis of anthocyanin by up-regulating the expression of Pb DFR,Pb ANS and Pb UFGT in pear peel.In addition,Y1 H and dual-luciferase assays showed that PbRVE1 b and PbRVE6 directly bound to the promoter of PbbHLH2 and activated its expression.The PbRVE1 b,PbRVE6 and PbbHLH2 transcription factors form a diurnal regulatory network of anthocyanin boisynthesis,which co-regulates the diurnal cycle of anthocyanin biosynthesis in pear.